The glycosyltransferases transfer a monosaccharide moiety of an activated sugar donor to an acceptor molecule, many requiring a metal ion cofactor. The majority of these enzymes are anchored in the Golgi compartment of eukaryotic cell as type II membrane proteins. They have a short N-terminal cytoplasmic domain, a membrane-spanning region, as well as a stem and a C-terminal catalytic domain that face the Golgi-lumen. The reaction catalyzed by these enzymes follows sequential ordered mechanism in which the metal ion and sugar-nucleotide bind first to the enzyme, followed by the acceptor. After the glycosyl moiety of the sugar-nucleotide donor is transferred to the acceptor, the saccharide product is ejected out, followed by the release of the nucleotide and the metal ion. The sugar transfer occurs either with the retention or inversion of the configuration at the anomeric carbon atom of the sugar donor. The structural studies on beta-1,4-galactosylransferase (b4Gal-T1), either free or complexed with substrates, first from our laboratory and later from other laboratories on other glycosyltransferases, have revealed that upon binding the donor substrate, one or two flexible loops undergo a marked conformational change, from an open to a closed conformation, simultaneously creating an acceptor binding site on the enzyme that did not exist before. Thus, the loop acts as a lid covering the bound donor substrate. After completion of the transfer of the glycosyl unit to the acceptor, the saccharide product is ejected; the loop reverts to its native conformation to release the remaining nucleotide moiety. In the case of b4Gal-T1, this conformational change also creates the binding site for cellular proteins like alpha-lactalbumin, Ovalbumin, former being a protein specific to mammary gland. The interaction of alpha-lactalbumin with galactosyltransferase enzyme changes the acceptor specificity of the enzyme towards glucose to synthesize lactose during lactation. Three important features have emerged from our studies: (I) Glycosyltransferases have flexible loop(s) which undergo conformational changes upon donor substrate binding and create the acceptor binding site. (II) In the metal-ion dependent glycosyltransferases, the binding site for the metal ion is located at the N-terminal hinge region of the flexible loop. (III) A few residues in the catalytic pocket determine the donor sugar specificity of the enzyme. Metal binding site located at the N-terminal hinge region of the long flexible loop is involved in the conformational change of the flexible loop: Crystallographic studies on the bovine b4Gal-T1 from our laboratory have shown that the primary metal binding site of the enzyme is located at the N-terminal hinge region of a long flexible loop, which upon binding of Mn2+ and UDP-Gal changes from an open to a closed conformation. This conformational change creates an oligosaccharide binding site in the enzyme. Neither UDP nor UDP analogues efficiently induce these conformational changes in the wild-type enzyme, thereby restricting the structural analysis of the acceptor binding site. The binding of Mn2+ involves an uncommon coordination to the S atom of Met344 residue; when it is mutated to His, the mutant M344H, in the presence of Mn2+ and UDP-hexanolamine, readily changes to closed conformation, facilitating the structural analysis of the enzyme bound with an oligosaccharide acceptor. Although the mutant M344H loses 98% of its Mn2+-dependent activity, it exhibits 25% of its activity in the presence of Mg2+ion. The enzyme kinetic studies with the mutant Met344His-Gal-T1 have shown that when Mn2+ is used during the catalytic cycle, at the product release phase the mutant enzyme is unable to return to the open conformation to release UDP and Mn2+.